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Universidad Autónoma de Madrid

Programa de doctorado en Biociencias Moleculares

Role of tetraspanin CD9 in murine experimental colitis

Doctoral Thesis

María Laura Saiz Álvarez

Madrid, 2018

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Departamento de Bioquímica Facultad de Medicina

Universidad Autónoma de Madrid

Programa de Doctorado en Biociencias Moleculares

Role of tetraspanin CD9 in murine experimental colitis

Memoria presentada por la licenciada en Bioquímica:

María Laura Saiz Álvarez

Para optar al título de Doctor por la Universidad Autónoma de Madrid Doctorado en Biociencias Moleculares

Director de tesis:

Dr. Francisco Sánchez-Madrid

Doctor en Ciencias Biológicas y Catedrático de Inmunología de la Universidad Autónoma de Madrid

Este trabajo se realizó en el Centro Nacional de Investigaciones Cardiovasculares (CNIC)

Madrid, 2018

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Francisco Sánchez Madrid, Doctor en Ciencias Biológicas y Catedrático de Inmunología de la Universidad Autónoma de Madrid,

CERTIFICA:

Que María Laura Saiz Álvarez, Licenciada en Bioquímica por la Universidad de Oviedo, ha realizado bajo su dirección el trabajo de investigación correspondiente a su Tesis Doctoral con el título:

Role of tetraspanin CD9 in murine experimental colitis

Revisado este trabajo, el que suscribe lo considera satisfactorio y autoriza su presentación para ser evaluado por el tribunal correspondiente.

Y para que así conste y a los efectos oportunos, firma el presente certificado en Madrid a 22 de mayo de 2018.

Fdo.: Prof. Francisco Sánchez-Madrid

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A mis padres, a Rocío y Luis, A mi abuela Carmen,

A Pablo.

“Hay una fuerza motriz más poderosa que el vapor, la electricidad y la energía atómica: la voluntad”

Albert Einstein (1879-1955)

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Agradecimientos

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Agradecimientos

En primer lugar, me gustaría darle las gracias a Paco por su apoyo y por haber sido un jefe excepcional tanto el ámbito profesional como personal. Gracias por haberme permitido aprender tanto en tu laboratorio, siempre formado por gente muy entusiasta e inteligente. Siempre recordaré las apasionadas discusiones en los seminarios. Gracias también por transmitirme esa perspectiva de sabio y por siempre sacar lo positivo de cada experimento. También gracias por compartir con tus estudiantes tus experiencias tan valiosas.

En segundo lugar, a Danay, sin ella nada de este trabajo hubiera sido posible. Gracias por tu apoyo desde el día uno, por ser tan buena profesora y transmitir todos tus conocimientos y razonamientos conmigo.

Gracias por tu paciencia con mi genética entropía, por enseñarme a razonar y por transmitirme tu rigor científico y la valía del esfuerzo. Un privilegio haber aprendido de tan gran científica, luchadora, apasionada e inconformista, de conocimiento insaciable. Gracias por tu franqueza, por intentar siempre sacar lo mejor de mí y enseñarme que en este mundo hay que hacerse valer. Y por supuesto gracias por las risas y por nuestros momentos “chocoboms”, ¡cuanto los echaré de menos!

Gracias a Olga, mi compañera de tesis todos estos años. Gracias por tu apoyo durante todo este complicado proceso y por tranquilizarme y comprenderme en los momentos más delicados. Gracias por preocuparte tanto por mí y sacarme de casa esos fines de semana de soledad en Madrid lejos de mi familia y amigos. Gracias por tu dulzura y cariño y por darme la fuerza que necesitaba para terminar esta dura etapa.

Cuánto echaré de menos esas meriendas pre-tésicas de risas y desahogo. Gracias Marta por esas largas y meditativas charlas donde siempre me hacías ver el lado positivo de las cosas y a estar agradecida con la vida.

Gracias por hablar siempre con esa franqueza que te caracteriza y tus valiosos consejos siempre acompañados de esa dosis de realidad que tanta falta nos hace a algunos de vez en cuando. Gracias también por las risas y por toda la ayuda con el proyecto. Me acordaré de ti cada vez que escuché la tabla de multiplicar del 4, y lo sabes jajaja. Gracias a Dani por las risas y esos momentos de desahogo existencial. Gracias a Raquelilla por su apoyo incondicional y preocupación el tiempo que hemos coincidido en el laboratorio, que, aunque corto, ha sido intenso. Qué gran compañera ha ganado el laboratorio de Paco con tu llegada, siempre con esa infinita disposición a ayudar a los demás y esa preocupación por que los experimentos salgan lo mejor posible. Gracias Anita por tu apoyo y tus sesiones de psicología. Gracias por escucharme en cualquier momento que necesitara.

Poca gente se pone en el lugar del otro como tú lo haces, gracias por tu sensibilidad y familiaridad. Gracias a Irene también tu sensibilidad, en eso Ana y tú os parecéis un rato, gracias también por tu apoyo y por sacarme del colapso cuando tenía que hacer algo relativo a la autónoma jaja. Gracias a Nieves, Lola, Noa y Eugenio por vuestro cariño y también apoyo. Gracias también a la gente que ya se ha ido. Gracias a Vera por sus consejos zen. Gracias a Noelia, Cris, Fran, Carol, Giulia, Dieguito y Jaso, con los que he compartido tantas experiencias y tantas charlas durante las comidas en el CNIC. Gracias a Rafa por ser tan buen amigo. Echaré de menos nuestros viajes matutinos hacia el CNIC. Gracias a Bárbara por tu apoyo desde que llegué a Madrid

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y acogerme con tanto cariño. Gracias por motivarme y animarme a continuar. Gracias también a Sheila y Myriam. Gracias a Helena por tu empatía, apoyo y comprensión durante toda la tesis. Gracias por esos fines de semana tan divertidos donde nos relajábamos y arreglábamos el mundo un poquito. Gracias por estar siempre pendiente de mí y de mi estado emocional, has sido un gran apoyo. Haber conocido a personas como tú hacen que la tesis haya merecido aún más la pena. Gracias también a los “Jacobs” en especial a Carlos, Esmeralda y Paula. Sois geniales. Gracias también a Mitchel. Gracias a Raquel Toribio. Gracias a José Pintor por los ánimos y por su apoyo. Esas conversaciones en el coche volviendo del CNIC a las tantas de la noche eran mejor que cualquier terapia jaja. Gracias Eva (María) por tu apoyo y por contagiarme tu alegría. Gracias por esas cenas en Bravo Murillo que tanto echaré de menos. Si hay algo bueno en haber alargado la tesis unos meses ha sido haber coincidido contigo y habernos visto tanto, como en los viejos tiempos. Gracias a Jesús también. Gracias a Coral, Marta y Bea. Aunque nuestra relación sea en la distancia sé que puedo contar con vosotras en cualquier momento. No faltaré a ninguna cena Navideña, lo prometo. Gracias a Coris, por todos estos años de amistad, por preocuparte tanto por mí y estar siempre ahí. Me llevo una joya de Madrid. Sin ti esta etapa no hubiera sido lo mismo y haces que mis recuerdos sean buenos y me olvide de los malos. Te admiro muchísimo. Gracias a Alberto también, ha sido todo un consuelo (a la par que un placer) compartir nuestras hipocondrías y dudas existenciales. Ya sabes, mal de muchos….

Gracias a las de siempre, mis amigas desde que tengo uso de razón de como dice Paco, mi “amada Salinas”. Gracias Lourdes, Estela, Ana, Cova, Zoraida, Paula, Belén y Candela. Aprovecho para pediros perdón si estos años no he estado tan pendiente de vosotras como me hubiera gustado, y gracias por entenderme cuando sabíais que tenía muy poco tiempo para cultivar nuestra amistad como se merece.

En especial quiero darle las gracias a mi familia y mi pareja. Su apoyo ha sido imprescindible. Gracias papá y mamá, por estar siempre ahí, por darme ánimos continuamente y el apoyo incondicional, por ayudarme también económicamente y entender mi situación y sobre todo por enseñarme la valía del esfuerzo. Sé que habéis sufrido conmigo esta etapa y un cachito de esta tesis os pertenece. Gracias a mis hermanos, Rocío y Luis, no se puede tener unos hermanos mejores. Gracias Rochi porque sé que a pesar de la distancia siempre has estado ahí. Gracias por las aliviantes charlas por skype o llamadas de whats ap en momentos de crisis.

Gracias por motivarme y empujarme hasta el final. Gracias a mi abuela Carmen, una mujer luchadora como ninguna, te admiro muchísimo. Gracias a mi prima Bárbara, que me contagia siempre esa energía que tiene y gracias a mis tíos Jesús y Patricia y también a mis primos Román y María, sois geniales. Gracias a Pablo, mi amigo y compañero de viaje, por cuidarme cada día, por animarme en cada paso que he dado y por soportar 7 años de relación en la distancia. Sin tu apoyo esto hubiera sido un imposible. Gracias por tu optimismo irrefrenable, tu motivación y cariño diarios. Por tu sentido del humor y por sacarme una sonrisa cada día después de un duro día en el laboratorio. Gracias por hacerme ver las cosas con la perspectiva que merecen y llevarme de la mano a tu nido de paz. Otro trocito de esta tesis también te pertenece a ti. Gracias también a Marisa y a Jose, por vuestro apoyo y acogimiento, para mi sois parte de mi familia.

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Summary

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Summary

Inflammatory bowel diseases (IBD), mainly consisting of Crohn’s disease (CD) and ulcerative colitis (UC) are chronic relapsing and remitting diseases resulting in uncontrolled inflammation of the gastrointestinal (GI) tract. The etiology of these diseases is unknown, but accumulating evidence suggests that it results from an inappropriate inflammatory response to intestinal microbes in a genetically susceptible host. The prevalence of IBD has increased during the last decades, overall in newly industrialized countries and is increasingly considered an emerging global disease. Unfortunately, IBD remains incurable due to its complex etiopathogenesis, and current treatments available only ameliorate symptoms. Current knowledge of these disorders are based on a combination of gene association studies, clinical investigations, and laboratory experiments in mice. The last ones have been very valuable tools to understand the pathogenesis of IBD.

In this thesis work, we investigated the role of CD9, a tetraspanin that regulates major biological processes such as cell migration and immunological responses, in two mouse models of colitis that have been used to study the pathogenesis of IBD. Tetraspanins are a family of proteins with four transmembrane domains that associate between themselves and cluster with other partner proteins, conforming a distinct class of membrane domains, the tetraspanin-enriched microdomains (TEMs). These TEMs constitute macromolecular signaling platforms that regulate key processes in several cellular settings controlling signaling thresholds and avidity of receptors. Previous in vitro studies revealed an important role for CD9 in the interaction of leukocytes with inflamed endothelium, but in vivo evidence of the involvement of this tetraspanin in inflammatory diseases is scarce. Here, we studied the role of CD9 in the pathogenesis of colitis in vivo. Colitis was induced by administration of dextran sodium sulfate (DSS), a chemical colitogen that causes epithelial disruption and intestinal inflammation. CD9-/- mice showed less severe colitis than wild-type counterparts upon exposure to DSS (2% solution) and enhanced survival in response to a lethal DSS dose (4%). Decreased neutrophil and macrophage cell infiltration was observed in colonic tissue from CD9-/- animals, in accordance with their lower serum levels of TNF-α, IL-6, and other proinflammatory cytokines in the colon. The specific role of CD9 in IBD was further dissected by transfer of CD4+ CD45RBhi naive T cells into the Rag1-/- mouse colitis model.

However, no significant differences were observed in these settings between both groups, ruling out a role for CD9 in IBD in the lymphoid compartment. Experiments with bone marrow chimeras revealed that CD9 in the non-hematopoietic compartment is involved in colon injury by limiting the proliferation of epithelial cells.

Future strategies to repress CD9 expression may be of therapeutic benefit in the treatment of IBD.

SUMMARY

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Resumen

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Resumen

La enfermedad inflamatoria intestinal (EII) engloba dos patologías, la colitis ulcerosa (CU) y la enfermedad de Crohn (EC), las cuales se caracterizan por una inflamación crónica intermitente de diferentes partes del tracto gastrointestinal. La etiología de la EII es desconocida, pero las evidencias obtenidas hasta ahora sugieren que son el resultado de una respuesta inflamatoria exacerbada desencadenada contra los microbios intestinales en sujetos con susceptibilidad genética. La prevalencia de la EII ha ido incrementando durante las últimas décadas, sobre todo en países recientemente industrializados alcanzando un carácter global.

Desafortunadamente, debido a la compleja etiopatogenia que subyace a la EII, ésta permanece incurable y los tratamientos actuales únicamente atenúan los síntomas. Los conocimientos que se tienen hoy en día acerca de estas enfermedades se basan en la combinación de estudios de asociación genética, estudios clínicos y experimentos con ratones en los laboratorios de investigación.

En este trabajo de tesis doctoral, hemos investigado el papel de CD9, una tetraspanina que regula importantes procesos biológicos tales como la migración celular o la respuesta inmunológica, en dos modelos murinos de colitis utilizados para estudiar la patogénesis de la EII. Las tetraspaninas son una familia de proteínas con cuatro dominios transmembrana que interaccionan entre sí y se agrupan con otras proteínas asociadas, conformando unos dominios de membrana, llamados microdominios enriquecidos en tetraspaninas o TEMs (del inglés, tetraspanin-enriched microdomains). Estos TEMs constituyen plataformas macromoleculares de señalización que regulan importantes procesos en diversos escenarios celulares controlando umbrales de señalización y avidez de receptores. Estudios previos in vitro han revelado un papel importante de CD9 en la interacción de los leucocitos con el endotelio inflamado, pero no existen prácticamente estudios in vivo relacionados con la implicación de esta tetraspanina en enfermedades inflamatorias. En este trabajo, estudiamos el papel de CD9 en la patogénesis de la colitis in vivo. La inducción de la colitis se realizó mediante la administración del químico colitogénico dextrano sulfato de sodio (DSS), el cual causa una disrupción de la barrera epitelial e inflamación intestinal. Los ratones CD9-/- mostraron menos colitis en comparación con los ratones de cepa salvaje tras la administración de una solución de DSS al 2% y una mayor supervivencia en respuesta a una dosis letal del químico al 4%. De manera concordante, se detectó una menor infiltración de neutrófilos y macrófagos en el colon de los ratones CD9-/-, así como una disminución de los niveles séricos de TNF-α, IL-6 y otras citoquinas pro-inflamatorias en el colon. El papel específico de CD9 en EII se investigó también en el modelo de transferencia de células T naïve CD4+CD45RBhi en animales Rag1-/-. Sin embargo, no se observaron diferencias entre ratones inyectados con linfocitos que expresaban CD9 o no. Los experimentos realizados en ratones quiméricos mediante trasplante de médula ósea demostraron que la no expresión de CD9 en el compartimento no hematopoyético era la responsable de mediar la protección frente a la colitis inducida por DSS. En estos animales se detectó una mayor proliferación del epitelio intestinal. Por ello, CD9 podría llegar a ser en un futuro una posible diana terapéutica para el tratamiento de la EII.

RESUMEN

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Index

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Agradecimientos Summary

Resumen Index

List of abbreviations ...

7

1. Introduction ...

11

1.1. Inflammatory bowel disease ... 11

1.1.1. Introduction ... 11

1.1.2. Epidemiology ... 12

1.1.3. Etiology: factors contributing to the development of intestinal inflammation ... 13

1.1.4. Mediators of intestinal inflammation... 15

1.1.5. Intestinal epithelial cell damage and mucosal repair ... 19

1.1.7. Mice models of intestinal inflammation ... 21

1.2. Tetraspanins ... 24

1.2.1. Introduction ... 24

1.2.2. Structure ... 24

1.2.3. Tetraspanin enriched microdomains (TEMs). ... 25

1.2.4. Tetraspanin CD9 ... 26

2. Objectives ...

35

3. Objetivos ...

39

4. Materials and methods ...

43

4.1. Mice ... 43

4.2. Genotyping of mice ... 43

4.3. Induction and assessment of DSS-induced colitis ... 44

4.4. T cell-mediated colitis ... 44

4.5. Bone marrow chimeras ... 44

4.6. In vivo permeability assay ... 45

4.7. Isolation and flow cytometry analysis of colonic leukocytes ... 45

4.8. Flow cytometric bead array (CBA) ... 45

4.9. RNA extraction and real-time quantitative PCR ... 45

4.10. In vitro T cell differentiation ... 47

4.11. Immunohistochemical analysis... 47

4.12. Statistical analysis ... 47

INDEX

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3

5. Results ...

51

5.1. CD9-/- mice are protected against DSS-induced colonic injury ... 51

5.2. CD9 exacerbates tissue injury and decreases mouse survival after a lethal DSS dose... 53

5.3. Reduced myeloid cell infiltration and proinflammatory cytokine expression in the colon of CD9-/- mice ... 54

5.4. CD9-/- bone marrow cells transplanted into WT mice do not provide protection against colonic injury ... 57

5.5. CD9 expression does not sensitize IECs to DSS-induced apoptosis ... 60

5.6. Enhanced colonocyte proliferation after DSS-induced injury in CD9-/- mice ... 60

5.7. Differential microbiota is not the cause of the decreased DSS susceptibility in CD9-/-mice... 63

5.8. CD9 expressed on CD4+ T cells does not contribute to immune-cell adoptive transfer-mediated colitis ... 64

5.9. T cell differential subset skewing in vitro shows no differences with CD9 expression ... 65

6. Discussion ...

71

6.1. CD9 and epithelial permeability ... 71

6.2. CD9 and inflammatory cell recruitment ... 73

6.3. CD9 and epithelial proliferation and apoptosis ... 75

6.4. CD9 and T cells ... 78

6.5. Future perspectives: IBD and CD9 ... 79

7. Conclusions ...

85

8. Conclusiones ...

89

9. References ...

93

10. Annexes ...

139

10.1 Publications related with this thesis work ... 139

10.2 Other publications ... 139

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List of abbreviations

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7

LIST OF ABBREVIATIONS

5-ASA: 5-aminosalicylic acid

6-MP : 6-mercaptopurine

ADAM: A Disintegrin and metalloproteinase domain-containing protein

AJ: Adherens junction

AJC: Apical junctional complex

ALCAM: Activated leukocyte cell adhesion molecule

AMPs: Antimicrobial peptides AOM: Azoxymethane

APCs: Antigen presenting cells AZA: Azathioprine

BMDCs: Bone marrow-derived dendritic cells BMP: Bone morphogenetic protein

CD: Crohn’s disease

CRC: Colorectal carcinoma

DRAP: Diphtheria toxin receptor-associated protein

DSS: Dextran sodium sulfate EC: Extracellular

ECM: Extracellular matrix EGF: Epidermal growth factor

EGFR: Epidermal growth factor receptor EIM: Extraintestinal manifestation

EO-IBD: Early-onset inflammatory bowel disease

EpCAM: Epithelial cell adhesion molecule ER: Endoplasmic reticulum

ERK: Extracellular signal-regulated kinase ERM: Ezrin/radixin/moesin

FAK: Focal adhesion kinase

FDA: Food and drug administration FITC: Fluorescein isothiocyanate FLIM: Fluorescence-lifetime imaging

FRET: Fluorescence Resonance Energy Transfer

GH: Growth hormone GI: Gastrointestinal

GM-CSF: Granulocyte-macrophage colony- stimulating factor

GPCR: G-protein-coupled receptor GPI: Glycophosphatidylinositol GST: Glutathione S-transferase

GWAS: Genome wide association studies HB-EGF: Heparin-binding EGF-like growth factor

HLA-DM: Human leukocyte antigen DM HNF4A: Hepatocyte nuclear factor 4 alpha HUVEC: Human endothelial umbilical cells IBD: Inflammatory bowel disease

IBS: Irritable bowel syndrome IC: Intracellular

ICAM-1: Intercellular adhesion molecule-1 IEC: Intestinal epithelial cell

IFN-γ: Interferon gamma IGF: Insulin-like growth factor

IRAK-M: Interleukin-1 receptor-associated kinase M

JAK: Janus kinase

JAM: Junctional adhesion molecule KGF: Keratinocyte growth factor KO: Knockout

LEL: Large extracellular loop

LFA-1: Lymphocyte function-associated antigen 1

LP: Lamina propria

LPA: Lysophosphatidic acid Mac-1: Macrophage-1 antigen

MAdCAM-1: Mucosal addresin cell adhesion molecule-1

MAPK: Mitogen activated protein kinase MCP-1: Monocyte chemoattractant protein-1 MH: Mucosal healing

LIST OF ABBREVIATIONS

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8 MHC-II: Major histocompatibility complex- II

MIP-1α/β: Macrophage inflammatory protein-1 alpha/beta

MMP: Matrix metalloproteinase MRP-1: Motility related protein-1 MTX: Methotrexate

NASH: Nonalcoholic fatty liver disease NETs: Neutrophil extracellular traps NF-kB: Nuclear factor kappa B NK: Natural killer

NLRP3: Nod-like receptor protein-3

NOD2: Nucleotide-binding oligomerization domain-containing protein 2

NSAIDs: Non-stereoidal anti-inflammatory drugs

PCD: Programmed cell death PDE-4: Phosphodiesterase-4

PI4-K: Phosphatidylinositol 4-kinase PKC: Protein kinase C

PMN: Polymorphonuclear

PRR: Pattern recognition receptor RA: Retinoic Acid

RNAi: Interfering RNA

SCFAs: Short chain fatty acids SEL: Short extracellular loop

SNPs: Single nucleotide polymorphisms TA: Transit amplifying

TCR: T-cell receptor

TEM: Tetraspanin enriched microdomain TFF: Trefoil factor

TGF-α: Transforming growth factor alpha TGF-β: Transforming growth factor beta TJ: Tight junction

TLR: Toll-like receptor TM: Transmembrane

TNF-α: Tumor necrosis factor alpha

TRAF6: TNF receptor associated factor 6 TREM2: Triggering receptor expressed on myeloid cells 2

TRIF: TIR-domain-containing adapter- inducing interferon-β

Tspan: Tetraspanin UC: Ulcerative colitis

VCAM-1: Vascular cell Adhesion molecule-1 VEO-IBD: Very-early-onset inflammatory bowel disease

VLA-4: Very late antigen-4

WAVE: Wiskott-Aldrich syndrome protein family verprolin-homologous protein

WGO: World gastroenterology organisation WT: Wild type

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Introduction

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1. INTRODUCTION

1.1. INFLAMMATORY BOWEL DISEASE

1.1.1. Introduction

Inflammatory bowel disease (IBD) defines a group of complex and multifactorial intestinal disorders, principally, ulcerative colitis (UC) and Crohn’s disease (CD). Both diseases are characterized by chronic inflammation of the gastrointestinal tract interspersed with relapsing phases [429]. UC and CD disorders have distinct pathological and clinical characteristics. CD affects any component of the gastrointestinal tract from the oral cavity to the annus (although it mostly associates with the terminal ileum and colon) in a non- continuous fashion, as opposed to a colon-limited disease in the case of UC (Figure 1.1). Transmural inflammation leading to a thickened colon wall and a typical “cobblestone” appearance are observed in CD, corresponding to longitudinal and circumferential fissures and ulcers that separate islands of mucosa (Figure 1.2) [298].

Moreover, CD can be associated with intestinal granulomas, strictures (narrowing of the intestinal lumen due to the scar tissue produced by the repair process after inflammation), and fistulas, but these are not typical findings in UC. On the other hand, UC involves superficial inflammatory changes, affecting mainly the mucosa and a thinner colon wall shows continuous inflammation with no patches of healthy tissue [3,560] (Figure 1.1), pseudopolyps (non-neoplastic lesions originating from the mucosa) (Figure 1.2), cryptitis and/or crypt abscesses. In both diseases, patients could experience diarrhea, fever and fatigue, abdominal pain and cramping, blood in stools, reduced appetite and weight loss [135]. The natural course of IBD alternates periods of remission and relapse, meaning that often IBD symptoms can be stable (low or absent) but suddenly worsen during a flare [429]. IBD diseases remain incurable and existing treatments only pale sympthoms and have a

Figure 1.1. Basic hallmarks and distribution of IBD disorders found in colon of patients with active disease. Left CD, right UC. Taken from MBBS medicine webpage.

INTRODUCTION

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limited efficiency [554]. Additionally, around 20-30% of UC and 30-40% of CD patients require surgical intervention at some point in their lifes to remove the affected region of the bowel [147]. Some of them may also require temporary or permanent colostomy or ileostomy. Nevertheless, in many cases, surgical resection or stomas do not achieve a permanent cure, and patients suffer relapses later in time [219]. On top of that, IBD patients display an elevated risk for colorectal carcinoma (CRC). In both cases, UC and CD, the risk of CRC is related to disease duration as well as the length of colon involved [125,126,232]. Thus, IBD constitutes an incurable, chronic, lifelong disease which entails detrimental consequences on patient’s quality of life [165]

and causes a major impact on health care resources [246,579].

1.1.2. Epidemiology

IBD could be diagnosed at any age, but the majority of new cases turn up in adolescence and early adulthood, being its peak onset in persons from 15 to 30 years of age [235]. Pediatric IBD (before 20 years old) accounts for approximately 25% of patients, with the peak onset in adolescence [466]. Nowadays, IBD affects around 5 million people worldwide, although the highest incidence and prevalence are found in westernized countries of Northern Europe and Northern America [9] (Table 1.1.)

However, during the last decades, both prevalence and incidence of IBD have increased in newly industrialized countries whose societies have become more westernized, evolving into a global disease [386,597]. Besides, economic advances inherent to industrialization increased health-care access and case reports. Importantly, Asia, South America and Middle East countries have documented the rise of IBD cases [388,454,474,528,563,586,638]. These cases report the same complications, morbidity, and mortality as in already settled patient populations in high-income countries [387,438]. Intriguingly, if epidemiological forecasts are correct, the global prevalence of IBD will affect tens of millions of people worldwide within a Figure 1.2. Typical endoscopic features of UC (B and C) and CD (D and E). (A) Normal looking mucosa in the descending colon (B) pseudopolyps (C) ulcers (D) cobblestone appearance (E) linear ulceration. Modified from: Lee JM. et al, 2016 [298].

A B C D E

Table 1.1. Highest annual incidence rates and reported prevalence rates for IBD. Taken from World Gastroenterology Organisation (WGO) webpage, data for 2015.

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few decades [243,244]. Along with industrialization, people from developing countries faced a series of changes in lifestyle behaviors that have been reported to correlate with IBD pathogenesis: 1) dietary changes from home-grown to processed foods which contain chemical food additives [98], reduction in fiber intake and breast-feeding [411]; 2) augmented antibiotic use [509]; 3) pollution exposure [35,404]; and 4) chronic stress [340], among others. Moreover, the rise in migratory rates to occidental countries impulsed by natural population increase, would also contribute to increase IBD incidence [37,306]. However, the exact causative relationships with IBD are yet to be unraveled and further research is needed to precisely elucidate the associated molecular alterations.

1.1.3. Etiology: factors contributing to the development of intestinal inflammation Although much progress has been made in understanding IBD, the exact etiopathogenesis remains unknown. Recent studies have implicated an interaction between genetics, immunology, environment, and microbiome [9].

Genetic factors

Accumulating evidence points out that IBD results from an inappropriate and continuing inflammatory response to intestinal microbes in a genetically susceptible individual [3], and a heritable component to the disease has been recognized [256]. Epidemiological evidence for a genetic contribution support this hypothesis [315]. Early epidemiological observations showed clear familial clustering in IBD, translated in higher risk ratios among first-degree relatives, especially siblings, and a positive family history [138,313,417]. Indeed, a positive family history is reported in around 8 to 12% of IBD patients [486]. Moreover, twin concordance rates are significantly greater in monozygotic than dizygotic twins for both CD (50-58% versus 0-12%) and UC (6- 14% versus 0-5%) [44,57,406,569], providing additional evidence for genetic contribution in IBD. Likewise, an increased burden of IBD has been observed in certain ethnic groups like Ashkenazi Jews, who present a significantly higher risk of developing CD irrespective of their geographical location [467,252]. As a complex multifactorial disorder, IBD possess the added difficulty that a susceptibility allele often requires other genetic and non-genetic cues to manifest its symptoms [256]. Thus, the genetic predisposition to develop IBD does not follow Mendelian inheritance patterns for a single locus, being instead polygenic in nature [86]. Only rare early-onset (EO-IBD) (before 5 years) and very-early-onset (VEO-IBD) (before 2 years) forms of IBD or IBD- like diseases (it is sometimes difficult to clearly ascribe the diseases to CD or UC), rely on monogenic defects [43,573]. Therefore, the advent of Genome Wide Association Studies (GWAS) at the beginning of the 21st century, constituted a crucial step towards the understanding of complex disorders like IBD. Importantly, the recent advances during the past two decades in sequencing techniques and the creation of large consortia assembling thousands of patients with IBD, allow for the discovery of many genetic risk loci, experiencing an exponential increase since GWAS development from 2005 onwards [110,614]. By now, around 200 genetic variants have been specifically found to be associated with CD, UC or both [81,345]. In fact, irrespective of their distinct clinical features, nearly 30% of IBD-related genetic loci are shared between CD and UC [256]

(Figure 1.3.). Analyses of the genetic risk loci implicated in IBD elucidated numerous pathways that are

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crucial for intestinal homeostasis. Genetic variations that disturb any stage of immune homeostasis, inflammation, or resolution can result in the exaggerated inflammatory response found in IBD [171]. Some of these genes are involved in epithelial barrier function, epithelial restitution, microbial defense, innate immune regulation, antigen sampling, antigen presentation or T cell differentiation, among others. At the cellular level,

processes like autophagy, endoplasmic reticulum (ER) stress, apoptosis, cell migration or oxidative stress could be also affected in IBD [256]. Genetic studies are already used to predict sensitivity to IBD therapies [55]. Combining these studies with different aspects of IBD pathophysiology will promote advances in translating genetic knowledge into predictive biomarkers and new treatments.

Environmental factors

Genetics alone cannot explain IBD. Allele variants found in genetic analyses can only explain about 20-25% of all IBD cases [315], and a lack of a 100% concordance rate in monozygotic twins is noticed. Thus, this is indicating microbiota and other environmental cues might be interacting with genetic susceptibility in the pathogenesis of the disease. In order to validate GWAS data, a number of genetically modified mice have been developed. However, a direct link of gene targeting to pathogenesis has not always been straight forward [77,108] supporting that additional environmental triggers are required for developing of the disease. The changing epidemiology of IBD across time and geography further supports environmental factors influence [9,245]. The more recent emergence of IBD in developing countries over the last two decades points to westernization of lifestyle and industrialization as the responsibles of introducing higher incidence numbers through environmental changes [597]. Some of these changes have been revised above, under “Epidemiology”

section. Mainly, drugs and antibiotics intake, infection, stress, smoking, pollution and changes in diet and intestinal microbial milieu (also affected through the use of antibiotics and infections) have all been implicated in the development of intestinal inflammation in cohorts of patients [10,291,339,455]. Besides, migratory data have been quite valuable and informative in this matter. Many migrant populations from developing low incidence countries acquire higher incidence rates than their country of origin and similar to the country they migrated to, mainly in the second generation [74,439].

Figure 1.3. Inflammatory bowel disease susceptibility loci. The loci are depicted by lead gene name, according to pathway. Loci attaining genome-wide significance (P<5x10-8) are shown for CD (red), UC (blue) and IBD (black). This last includes the shared loci that confer susceptibility to both CD and UC (approximately one-third of loci). From Lees CW. et al., 2011[299].

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15 Gut microbiota in IBD

The human GI tract harbors a complex microbial community containing over 1014 microorganisms of greater than 1000 different species, which occupy a vast mucosal interface (250-400m2) of the human body [22]. As a result of many years of co-evolution, the host immune system has been shaped to tolerate commensals as a means to maintain a symbiotic relationship within the human body [20,104]. Some of the gut microbiota benefits include nutrient metabolism, xenobiotic and drug metabolism, maintenance of structural integrity of the gut mucosal barrier, immunomodulation, and protection against colonization of pathogenic microorganisms [229]. Tolerance mechanisms exerted by microbial species include inhibition of proinflammatory cytokines [325,50] and NF-κB induction [616], compartmentalization of mucosal IgA secretion [326], promotion of regulatory T cells (Tregs) generation [158,446,468,367], secretion of anti- inflammatory cytokines like IL-10 [185,51] and generation of metabolic substrates that stimulate mucosal barrier functions, like butyrate [459]. In IBD, microbiota can act as a double edged-sword, as it is important in both maintaining health and mediating disease [324]. On one hand, it is indispensable for colitis development [174,488,103,536] and, for instance, IL10KO mice housed in germ-free facilities do not develop colitis at all, as opposed to their counterparts raised under conventional conditions [284]. The clinical observation that IBD can respond to antibiotic treatment substantiate that gut microbiota is an essential factor in driving the inflammatory response [593,254,75]. Moreover, the detection of more inflammation associated to certain anatomical regions like the terminal ileum and rectum with faecal stasis [270], together with inflammatory remission observed in the excluded intestinal segment after faecal diversion (by means of an ileostomy or colostomy) [230], and reactivation after reinfusion of intestinal contents [105], support this notion. On the other hand, certain immunoregulatory bacterial subpopulations help to maintain intestinal homeostasis [240].

On top of this, we have to take into account that several aspects may complex the study of the relationship between microbiota and IBD. First, there is a huge interindividual variability (even among monozygotic twins only the 40% of faecal phylotypes are shared) [567], secondly, the vast majority of studies are performed in mice whose microbiota composition can be finely tuned and not always a correlation with humans occurs [389], and lastly, it is sometimes difficult to ascribe whether the changes detected on microbial populations are cause or consequence of enterocolitis [390].

1.1.4. Mediators of intestinal inflammation

The intestinal mucosa is constantly exposed to commensal bacteria and dietary antigens. The first line of defense is constituted by the intestinal epithelial cell line, which provides a physical barrier to pathogens but also acts as a frontline sensor that translates information to underneath mucosal immune cells. Thus, epithelial barrier disruption accounts for IBD pathogenesis, and alterations in these cells functions have been found in IBD patients [420]. In a normal situation, after pathogen clearance, resolution of the inflammatory process is required to fully achieve the restoration of tissue integrity and function [407]. Unfortunately, may the host have a genetic susceptibility trait and/or failure to accurately trigger tolerance mechanisms, an acute

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inflammatory response that should be dampened could derive in chronic inflammation, leading to IBD [464,487].

Disruption of the epithelial cell barrier

Intestinal epithelial cells (IECs) have a dual role, regulating water and nutrients uptake while at the same time acting as a barrier to the permeation of luminal pathogens and potentially harmful substances. The GI columnar epithelium is covered by a single-cell-thick layer, composed of different subtypes of specialized IECs, including absorptive cells (the most abundant, up to 80% of all epithelial cells), goblet cells, enteroendocrine cells, Paneth cells (only found in small intestine), M cells and Tuft cells [420], all of which differentiate from epithelial stem cells [392]. Of note, mucus production by globet cells is of vital importance for keeping at bay luminal microbes [233,234], emphasized by observations in active enterocolitis of an altered thickness, continuity [442] and composition [58,88] of the intestinal mucus layer, as well as an altered mucin structure [293,79]. Moreover, globet cell and mucus depletion is characteristic of UC [231] and Muc2 knockout mice develop spontaneous colitis [578]. Paneth cells are specialized for the secretion of many antimicrobial peptides (AMPs), including defensins (cryptdins in mice), cathelicidins and lysozyme; representing key players in innate immunity in the gut. Indeed, a reduced expression of α-defensin mRNA has been detected in ileal biopsies from CD patients [596] and Paneth cell abnormalities were found to be associated with mucosal dysbiosis in the context of CD [314].

An effective sealing and proper paracellular permeability is achieved thanks to the apical junctional complex (AJC), located at the enterocyte apical pole, and composed by tight junction (TJ) and subjacent adherens junction (AJ) protein complexes. Both TJ and AJ are reinforced by a dense perijunctional ring of actin and myosin [568]. TJs consist of the transmembrane proteins occludin, the claudin family of proteins and junctional adhesion molecules (JAMs), and its cytoplasmic plaque involves mainly the scaffolding zonula occludens family of proteins and cingulin [566]. AJs together with desmosomes, provide the adhesive strength required for maintenance of cell-cell interactions, whereas TJs are the responsible for sealing the paracellular space and also act as fence to prevent the mixing of membrane proteins between the apical and basolateral membranes, thus contributing to epithelial cell polarization [397]. In the setting of IBD, a compromised integrity of the epithelial barrier and an increased permeability have been recognized since the eighties, in both ulcerated and non-ulcerated epithelia [574,415,200] and in first-degree relatives [525,501,202]. Latter studies revealed altered TJ function, ultrastructure and protein composition in IBD patients [437,499] and reduced expression of occludin [281] as well as the AJ molecules E-cadherin and α-catenin in both CD and UC [120,247].

However, it is not known for certain whether impaired barrier function is secondary to gut inflammation and tissue damage, or if it is a self-determining event. Evidence suggesting prior barrier defects contributing to IBD come from reports documenting changes in intestinal permeability 8 years before onset of CD [224], increased intestinal permeability in 10-54% of first-degree relatives in the absence of clinical symptoms [416,341,201] and animal models of IBD that documented increased epithelial permeability prior to the inflammatory process [192]. In this regard, intestinal permeability has been reported to be a trustable

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prognostic indicator of relapse to active disease in patients with CD during clinical remission [106,610]. In favor of a consequence rather than a cause, proinflammatory cytokines produced in excess in IBD like IFN-γ, TNF-α, and IL-13 can lead to cytoskeletal rearrangements that disrupt TJ and increase the epithelial permeability further aggravating inflammation in active disease [478,333,329,46]. Moreover, some experiments in transgenic mice with genetic defects in TJ associated proteins suggest that disruption of barrier function alone is not always enough to trigger disease [538,257]. Overall, intestinal barrier loss can be considered as one component that contributes to a multifactorial mechanism of IBD pathogenesis [398].

Mucosal immune response dysregulation

IECs do not regulate intestinal homeostasis by their own, but instead establish a dynamic crosstalk with intestinal microbes and local immune cell populations. Microbial cells in the intestinal lumen are estimated to outnumber our own somatic and germ cells by one order of magnitude (~1014) [444]. Thus, a sophisticated immune system in our GI tract, shaped after millions of years of co-evolution, is tasked to remain tolerant to this huge quantity of beneficial commensal microorganisms, while accurately prevents the invasion of occasionally incoming pathogens.

Innate immune responses

Innate immune cells from the intestinal mucosa can monitor microbial ligands using PRRs, and microbial metabolites using G-protein-coupled receptors (GPCRs) and solute carriers. Unlike other tissues, in the gut, incoming monocytes are continuously replenishing the macrophage pool in the intestine [23].

Interestingly, depending on the context, the same Ly6Chi monocyte precursors can give rise to hyporesponsive macrophages in homeostasis or proinflammatory macrophages in an inflammatory context [24]. In a resting healthy epithelium, gut-resident macrophages have a limited capacity to respond to bacterial adjuvants, despite they express a full repertoire of TLR receptors. In this sense, downregulation of its downstream mediators including MyD88, TRAF6, TRIF and CD14 [524,523,522] and/or increased expression of molecules that impair TLR signalling like IRAK-M and IkBNS [198], are responsible for their hyporesponsiveness. Similarly, IECs typically have low levels of TLRs, and some of these are exclusively located in the basolateral membrane of the cell, which allow epithelial cells to reside in the high bacterial concentration of the distal ileum and colon and respond only when the epithelial cell line is broken [629]. In intestinal DCs, the hyporesponsiveness is thought to be restricted to TLR4 [78]. Importantly, intestinal CD103+ DCs have the unique ability to induce the differentiation of peripheral Foxp3+ T regulatory (Treg) cells from naïve T cells in a process dependent on retinoic acid (RA) and active transforming growth factor (TGF)-β [540,85], thus playing a key role in the development of intestinal tolerance. In an inflammatory context, macrophages and DCs in the lamina propria (LP) are increased in absolute numbers and have an activated phenotype in both forms of IBD [487], mainly derived from incoming inflammatory monocytes that migrate to the inflammatory focus. These monocytes conserved normal TLR levels, as polymorphonuclear cells do. Thus, an extensive array of proinflammatory cytokines mainly IL1-β, TNF-α, IL-6, IL-8, members of the IL-12 family [382] and chemokines like MCP-1, MIP-1α, MIP-1β, RANTES, CXCL5 and fractalkine (CX3CL1), were all described to be augmented in IBD [483,572,173]. The selective inhibition of all these proinflammatory mediators has been proved to attenuate

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the onset of experimental colitis [487]. On the other hand, accumulating evidence claims for deficient innate immune responses in CD. Increased CD susceptibility associated with NOD2 polymorphisms [218] (which encodes the receptor of the muramyl dipeptide bacterial wall molecule) and the therapeutic activity of granulocyte-macrophage colony-stimulating factor (GM-CSF) [273] support the hypothesis that CD is the result of defective bacterial killing. In this regard, several studies have reported neutrophil and/or macrophage dysfunction in CD patients [520,582,184,503] that would imply a deficient removal of bacterial antigens leading to the subsequent chronic inflammatory status [150]. However, in UC, neutrophil over-activation may imply collateral tissue damage as the extent of neutrophil infiltration correlates with the severity of the disease [60] and neutrophil chemotaxis and ROS production are both increased [502,449].

Adaptive immune responses

The traditional concept over the past decade that in CD Th1 and Th17 responses are selectively upregulated but not in UC, which was thought to represent a Th2-driven disease, has been challenged [112].

Mucosal levels of typical Th1 cytokines like IL-12, IL-18, IL-2, TNF-α and IFNγ are increased in CD patients [393,59,359,360] whereas numerous reports describe an atypical Th2 response in UC, mediated by natural killer (NK) T cells that secrete IL-13 [187,159], although the upregulation of IL-4 and IL-5 Th2 cytokines is variable in UC tissues [160]. On the other hand, an upregulation of prototypic Th2 cytokines like IL-5 and IL- 13, was observed in an animal model of human CD [27] and also of IL-4 and IL-5 in lesions of patients with CD [27]. Moreover, the efficacy of anti TNF-α treatment in dampening Th1 responses on UC patients [472], suggests that this T helper subset also plays an important role in UC. The production of IL-17 is stimulated by the production of IL-6, TGF-β and IL-23 by innate immune cells and APCs, especially dendritic cells. Levels of both IL-23 and IL-17 are increased in CD tissues and several types of experimental colitis [626,497,154].

Despite of that, administration of anti-IL-17A monoclonal antibody to patients with moderate to severe CD had no therapeutic effect and in some cases exacerbated the disease [217]. Moreover, anti-IL23p19 therapy failed to meet clinical remission end points [482]. Tregs are crucially involved in the maintenance of gut mucosal homeostasis by suppressing abnormal immune responses against commensal flora or dietary antigens.

They have a potent anti-inflammatory role in experimental colitis [515,136], and also are depleted in peripheral blood of patients with active IBD [92].

Leukocyte extravasation

The steps of the leukocyte extravasation cascade comprise the sequential adhesion steps known as tethering, rolling, tight adhesion and transmigration [394]. Inflammatory cytokines upregulate local endothelial expression of adhesion molecules that cause circulating leukocytes to adhere to the inflamed endothelium [30].

Tethering and rolling are mediated by selectins and their carbohydrate ligands [304]. The selectins are a family of transmembrane mammalian lectins expressed on the surface of leukocytes (L-selectin), endothelial cells (P- , E-selectins), and platelets (P-selectin) [242]. The importance of each selectin varies between organs and the inflammatory stimuli [304]. In animal models with intestinal inflammation, conflicting results were obtained as double deficient mice for E and P-selectins showed enhanced leukocyte recruitment and more severe disease [342], and no protection was observed after blocking neither E-selectin nor P-selectin or L-selectin [484];

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whereas others observed colitis severity attenuation by P-selectin blocking antibodies or in P-selectin deficient mice [627,588,168]. Nevertheless, more robust effects were observed with the blockade of tight adhesion molecules like Intercellular adhesion molecule-1 (ICAM-1) or CD54, Vascular Cell Adhesion Molecule-1 (VCAM-1) or CD106, and Mucosal Addressin Cell Adhesion Molecule-1 (MAdCAM-1), all playing important roles in the dysregulated trafficking of leukocytes that occurs during IBD [83,62,107,237] and in mice models of intestinal inflammation [250,249,541]. Circulating T cells that bear the gut-homing α4β7 integrin bind to the MAdCAM expressing colonic endothelium [42], which increases its expression levels upon inflammation [83]. Likewise, VCAM-1 and ICAM-1 protein levels are rapidly upregulated in an inflammatory onset [124,72]. VCAM-1 is the ligand for α4β1 (VLA-4) [132] and can also bind α4β7 albeit with lower affinity [465]. ICAM-1 is the ligand for LFA-1 (αLβ2) [336] and Mac-1 (αMβ2) [114]. Thus, blockade of these adhesion molecules have proven efficacy in acute colitis models in mice and rats [38,182,553,608,137,627]

and in human clinical trials [580]. On the leukocyte side, targeted blockade of a combinatorial epitope on the α4β7 integrins in cotton-top tamarins [193], in mice [422] and humans [140], and of β2 integrin (CD18) in rats [346] were successful in attenuating colitis. The selective expression of CCL25 by colonic endothelium mediates the gut-specific recruitment of T cells that express the chemokine receptor CCR9 [208]. However, during intestinal inflammation, CCR2 expressing CD4+ T lymphocytes are preferentially recruited to the inflamed ileum [84], and an amelioration of colitis was observed in monocytopenic CCR2-deficient mice [427]

and in mice depleted of CCR2-expressing cells [643].

1.1.5. Intestinal epithelial cell damage and mucosal repair

Cell death is an integral part of tissue homeostasis. Elimination of damage or aged cells through programmed cell death (PCD) by apoptosis or autophagy processes is of particular importance for the GI tract, which is continuously exposed to environmental harmful agents [380]. However, in some settings, immune- cell-derived triggers, cytotoxic drugs and physical stressors can lead to excessive IEC apoptosis and undiscriminating necrosis, causing extensive tissue damage. Upon infection, intracellular virus and bacteria induce host cell death through several distinct modalities, including apoptosis, necrosis, and inflammasome- mediated pyroptosis [300,19]. Besides, activation of macrophages and T cells leads to TNF and FasL expression, which promotes apoptosis in mature epithelial cells and crypt cells [348,177,302]. Other proinflammatory cytokines like IFN-γ [471,177] or IL-12 [177] can also trigger epithelial apoptosis. Of note, the intestinal epithelium is much more sensitive to the apoptosis-inducing activity of TNF-α than other tissues [238,424] and apart of inducing apoptosis leads to the disruption of TJ [625], further fueling inflammation and tissue destruction. Neutrophil serine proteases such as elastase, proteinase-3 and cathepsin G, and matrix metalloproteinases like MMP-9, also contribute to further amplify tissue damage due to the relatively indiscriminate range of action of these molecules in terms of their target [280]. Apart from infections, other environmental agents can cause a disruption of the epithelial cell barrier. Ionizing radiation [221] and a variety of drugs like non-stereoidal anti-inflammatory drugs (NSAIDs) [606] or chemotherapy agents [433], provoke apoptosis of IECs. Indeed, many of these molecules are causative agents of IBD development or relapses

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[271,550]. After epithelial injury and barrier breakdown, signals from nearby IECs, underlying stromal cells and immune cells promote rapid resealing of the epithelial cell lining to reestablish homeostasis. In a simplified way, epithelial repair is achieved by three distinct mechanisms: restitution, proliferation and differentiation [117].

Epithelial restitution

Despite the need for an impenetrable defence, the intestinal epithelium is only one cell thick to allow for the exchange of nutrients and solutes. Thus, the integrity of the intestinal barrier can be easily compromised, and mechanisms or rapid gap sealing are required. To accomplish this, the first step in mucosal healing consist on a quick migration to the denuded area of cells surrounding the wound, which is called epithelial restitution.

This process starts within minutes to hours of injury and is independent of proliferation [555,119]. During restitution, epithelial cells lose their columnar polarity and experience and extensive actin cytoskeletal rearrangement, mediated by Rho family of GTPases [485,204]. Cells lose their microvilli and apical/basolateral orientation, flatten, and extend their lamellipodia into the denudated area [63,287]. New focal attachments are formed at the leading edge mediated by integrins engagement [539,320,319] with clustering of focal adhesion kinase (FAK) [544,628] and where extracellular matrix (ECM) proteins play a key role [458,169,33]. GI restitution is modulated by several factors including: 1) growth factors [100,183,391,489,148,571,119,601,430]; 2) cytokines such as IL-2, IFN-γ and IL-1β [70,119]; 3) chemokines CXCL12 and CCL20 [587,521]; 4) prostaglandins [648] and 5) other luminal factors like short chain fatty acids (SCFAs) [600], bile acids [290], polyamines [453,591], lysophosphatidic acid (LPA) [197] and trefoil factors (TFF) [199].

Epithelial proliferation

After restitution, cell proliferation is triggered to increase the pool of enterocytes available to resurface the denudated area, generally beginning hours to days after injury [310]. Stem cells located at the base of the crypts experience asymmetric cell division, generating one rapidly cycling daughter cell, while the other daughter cell replaces the parent stem cell. The more rapidly proliferating daughter cells, also called transit- amplifying (TA) cells, are the responsible for building tissue mass, and undergo a limited number of cell divisions before terminally differentiating into the functional cells of the tissue [29]. The proliferation phase is temporally and spatially distinct from migration, and at the molecular level these processes depend on excluding patterns of kinase activation and gene expression. For instance, EGF promotes p38 mitogen- activated protein kinase (MAPK) migration or ERK-dependent proliferation, which oppose one another’s function [152]. TGF-β [264], LPA [537] and adenine nucleotides [118] stimulate migration, but blunt proliferation. In vivo, rapid and immediate migration of injured epithelia is followed by a surge of proliferation much later in time, supporting temporal disassociation of migration and proliferation [287,144,143]. Several molecules modulating intestinal epithelial proliferation are peptide growth factors, such as EGF, TGF-α, and insulin-like growth factor 1 (IGF-1) [323]; peptide hormones like neurotensin, cholecystokinin, bombesin, peptide YY [556,170] and glucagon-like peptide 2 [121]; prostaglandins [557]; cytokines like IL-6 [283], IL- 22 [423] and IL-10 [447] and TLR agonists, among the most important ones. TLRs activation in IECs promotes

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the proliferation of epithelial stem cells by inducing the production of ligands for EGFR, such as amphiregulin and prostaglandin E2 [211,155]. Also, changes in ECM elasticity after intestinal damage promote the new formation of FAK-integrin complexes, cyclin D1 upregulation and progression through the cell cycle [412].

Importantly, type 2 immunity responses promote epithelial cell proliferation, in a way to favor worm expulsion and initiate the repair process of the damaged barrier [441]. In this sense, type 2 cytokine IL-4 [344] promotes proliferation acting directly on the IEC and indirectly, together with IL-13, induce the expression of the cell surface receptor Trem2 (with as yet undetermined ligand) on M2 macrophages, which have a critical role in the proliferative, but not in the migratory phase, of intestinal wound repair [506].

Epithelial maturation and differentiation.

Once the gap has been filled, epithelial maturation and differentiation is required to restore villus architecture as well as digestive, absorptive and defensive functions. The signaling pathways and transcription factors implicated in the regulation of cell fate determination and lineage specific differentiation in the intestine are becoming increasingly well understood. Among these molecular pathways we can find Wnt-β-catenin-TCF [334,272], Notch and its downstream effectors HES1 and Math1 [620], BMP-TGF-β-SMAD [186,34] and hedgehog signaling [576]. Moreover, E-cadherin-mediated cell-cell [500] and integrin-mediated cell-ECM adhesion [330], and an array of cytokines, hormones and growth factors, are also involved in ruling IECs maturation [251]. Noteworthy, imbalances in these processes ranging from death mechanisms of IECs to the recovery mechanisms including epithelial restitution, proliferation and maturation have been involved in IBD pathogenesis.

1.1.7. Mice models of intestinal inflammation

The development of murine models of acute and chronic intestinal inflammation have provided valuable insights into the complex mechanisms behind IBD pathogenesis. Mice models of IBD can be classified as spontaneous or inducible through chemicals, bacterial infection, immune cells adoptive transfer, transgenesis and gene depletion (Table 1.2.).

Table 1.2. Mice models of intestinal inflammation

Spontaneous Chemically

induced Infection Adoptive transfer Transgenic Knockout

C3H/HeJBir [562]

Samp1/Yit[282]

DSS [415]

TNBS/DNBS[398,188]

Oxazolone[51]

Acetic acid[338]

Carrageenan[141]

Indomethacin [638]

Peptidoglycan- polysaccharide[471]

Salmonella- induced[185]

Citrobacter rodentium[200]

Adherent invasive E.coli[331]

CD45RBhi  Rag1-/- /SCID[377,451]

BM  Cd3εTg26[207]

Hsp60 CD8+  TCRβ-/-[551]

STAT-4[628]

dn-N- cadherin[195]

IL-7[617]

IL-2[490]

MUC2[600]

IL-10[42]

STAT3[569]

XBP1[254]

CRF2-4[550]

TGF-β[531]

Giα2[487]

TCR-α[372]

A20[308]

NEMO[394]

MDR1A[428]

TNFΔARE[279]

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22 DSS colitis model

Dextran sodium sulfate (DSS)-induced colitis has become a widely used model for studying IBD in the mouse [87,401] and among the chemically induced colitis models, is one of the most used due to its rapid development, highly reproducibility and easy way of administration (by water intake). Moreover, acute or chronic models of intestinal inflammation can be achieved by modifying the concentration of DSS and the frequency of administration. Acute colitis is usually induced by continuous administration of 2-5% DSS for short period (5-9 days), whereas chronic colitis may be generated by continuous treatment of low concentrations of DSS or cyclical administration of higher concentrations of DSS intercalated with water intake [131]. Each version is used to study innate vs adaptive immune responses, respectively. This model is suitable for studying events triggered by temporary failure of mucosal homeostasis after epithelial cell shedding and loss of barrier integrity, and can also provide insight into the mechanisms that lead to mucosal healing after initial injury [258]. DSS is an anionic surfactant that disrupts the epithelial cell monolayer lining, leading to the entry of luminal bacteria and associated antigens and stimulating local inflammation [97].

Several mechanisms have been postulated about DSS-induced colonic mucosal inflammation. Recent results indicate that sulfate groups of the DSS molecules destabilize the mucus layers and make them more permeable to bacteria [421]. Moreover, it also exerts a direct toxic effect on the epithelial cells due to its surfactant properties and fusion with colonocyte membranes through the formation of nano-lipocomplexes with medium- chain-length fatty acids (MCFAs) in the colon [292].

Figure 1.4. Schematic progression of DSS-induced colonic damage. Control colon shows straight healthy crypts with their base sitting on the muscularis mucosae and plenty of big globet cells. As the damage goes on, globet cell depletion and crypts shortening are detected. Grade 1 lesion: loss of the basal one-third of the crypts, which fails to sit on the muscularis mucosae. No inflammation can be appreciated yet. Grade 2 lesion: loss of the basal two-thirds and focal thinning of the epithelium. Some inflammation in the lamina propria is beginning to be appreciated. Grade 3 lesion: Loss of the entire crypt with retainment of the surface epithelium. LP and submucosa show broader inflammatory infiltrate.

Grade 4 lesion: Total disappearance of the epithelial cell lining and huge inflammatory infiltrate. Modified from Cooper HS. et al, 1993 [87].

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Interestingly, DSS-induced extensive pathology is mainly confined to the large intestine, specifically the distal colon, where an enormous number of microorganisms live and where the absorption of MCFAs takes place [97]. DSS administration induces changes in the expression of tight junction proteins [432] and increased expression of proinflammatory cytokines [617] as soon as day 1 after treatment. These modest initial effects are followed by increasingly drastic symptoms. During DSS administration, mice can exhibit pronounced weight loss, increased intestinal permeability, diarrhea, severe bleeding, hunched back, piloerection, anaemia and eventually death. The typical histological changes induced by acute DSS include mucin and globet cell depletion, epithelial erosion, ulceration, submucosa swelling and a mixed inflammatory infiltrate of neutrophils and macrophages in the LP and submucosa [419] (Figure 1.4) . Importantly, DSS-induced colitis model responds to typical treatments of human disease [347], thus represents a relevant model for the translation of data from mice to humans.

Adoptive transfer colitis model

Transfer of naïve (CD45RBhi) CD4+ T cells into syngeneic immunodeficient (lymphopenic) SCID or Rag1-/- recipients leads to wasting disease and colitis [436,364]. Intestinal inflammation develops after 5 to 10 weeks after treatment. As lymphopenic recipient mice do not have any T cell population, nor Treg cells, they cannot dampen the expansion of naïve T cells into the Th1 and Th17 proinflammatory T cell populations that differentiate after several weeks in the intestine due to commensal microbiota stimulation. In this line, the transfer of mature CD4+CD45RBlow (which include Tregs) [435] or directly the transfer CD4+CD25+ Tregs [366] inhibits the induction of the disease. Macroscopically, mice inflamed colons are thickened and shortened compared to non-transferred control mice. Histopathological inspection of distal colon obtained from mice with active disease reveals a typical transmural inflammation, epithelial cell hyperplasia, polymorphonuclear leukocyte (PMN) and mononuclear leukocyte infiltration, crypt abscesses, and epithelial cell erosions [409]

(Figure 1.5). This model is very useful for studying the mechanisms that govern Th1 and Th17 T-cell differentiation, the role by Tregs in suppressing or limiting intestinal inflammation, as other immunoregulatory promoters like some bacterial species [258]. Thus, this model helped to define and establish the basis of mucosal homeostasis dependence on a balance between proinflammatory effector and anti-inflammatory regulatory functions.

Figure 1.5. Histological magnification of colon obtained from RAG-/- at 8 weeks following adoptive transfer with CD4+CD45RBhigh T cells. A large mixed leukocyte infiltrate composed primarily of granulocytes, T cells, and monocytes (inset) can be detected. Increased bowel thickness and transmural injury affecting all layers of the colon including loss of globet cells, epithelial disruption and erosion and even muscularis mucosae distortion are observed.

From Ostantin DV. et al, 2008 [409].

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